Immunofluorescence of wild type and mutant GLUT1
After transfection with the plasmids, GLUT1 was detected with anti-myc and anti-mouse IgG Alexa Fluor 594, as secondary antibody. Immunofluorescent microscopy revealed localization of GLUT1 at the cell membrane of GLUT1-wild type, A405D, and R333W (white arrow). GLUT1 was not detected in SLC2A1V303fs transfected cells. Additionally, V303fs without stop codon was faintly stained in the cytoplasm, but not stained in the cell membrane (yellow arrow). Hoechst 33342 was applied to stain nuclei.

Rota-rod test after AAV-hSLC2A1 injection.
The mice were trained in acceleration mode from 4 to 40 rpm 3 times daily for 2 continuous days. At 30 min after the final training session on the second day, the mice were tested. The bar graph shows the time taken to fall off the rotating drum during the final test. The Rota-rod test was performed at the age of 10 and 14 weeks, corresponding to 4 and 8 weeks after intra-cerebroventricular injection, respectively. There was a significant deference between Wt (GLUT1^+/+) mice and control GLUT1^+/- mice (GLUT1^+/- mice after intra-cerebroventricular injection with saline) (P < 0.05). In both injection groups, the Rota-rod test showed trends for motor function improvement compared with control GLUT1^+/- mice. Especially, at 4 weeks after intra-cerebroventricular injection of the untagged AAV-hSLC2A1 group, motor function was significantly improved (P < 0.05). *P < 0.05. Bar height; mean ± SE. Wt: GLUT1^+/+ mice; Ht: GLUT1^+/- mice; icv: intra-cerebroventricular injection; ip: intraperitoneal injection; Ht-AAV (no tag) icv: GLUT1^+/- mice after intra-cerebroventricular injection of untagged AAV-hSLC2A1.

Types of cells in brain tissue that are stained by an anti-myc antibody at 6 months after direct CNS administration of AAV-GLUT1.
Exogenous GLUT1 (GLUT1-myc) expression was detected with an anti-myc antibody (a–d), and myc-immunoreactive cells were co-stained using anti-CD31 (endothelial marker; e), anti-beta III tubulin (neuron-specific marker; f), anti-GFAP (astrocyte marker; g), and anti-PLP1 (oligodendrocyte marker; h) antibodies in the same manner as in Fig. 6. Myc-immunoreactive cells were mainly co-stained by the anti-CD31 antibody (i), and some of the myc-immunoreactive cells were co-stained by the anti-beta-III tubulin and PLP1 antibodies (j, l). No immunofluorescence was detected with the anti-GFAP antibody (k).